The in vitro cytotoxic response of human dermal and colon cell lines to structurally well-defined full generation cationic dendritic polyamidoamine (PAMAM) nanoparticles of generations G4, G5, G6 was investigated. PAMAM dendrimers have been demonstrated to elicit a well defined cytotoxicological response from Alamar Blue, Neutral Red and MTT assays, where the response increases systematically with dendrimer generation and number of surface amino groups. A good correlation was found between the EC50 values of these assays. This systematic response is furthermore demonstrated for the generation of reactive oxygen species, mitochondrial membrane potential, inflammatory responses, lysosomal activity, caspase activation, apoptosis and DNA damage. The mechanism of endosomal escape of PAMAM by the so-called ‘proton-sponge effect’ was also studied. The results are consistent with a pathway of endosomal uptake of PAMAM, followed by endosomal rupture and subsequent localisation of PAMAM dendrimers in the mitochondria, leading to PAMAM generation, dose and time dependant biphasic ROS production and caspase- 8 and 3 activation, inflammatory responses (TNF-α, IL-6 and IL-8 expression), apoptosis and DNA damage. Overall, significant differences are observed between the responses of the dermal and colon cell lines, and it is suggested that these can be understood in terms of differing intrinsic antioxidant levels.